ES2252009T3 - LICOPENO PRODUCTION METHOD. - Google Patents

Abstract

Process for the production of lycopene. The present invention refers to a new process of obtaining carotenoids, especially such as Blakeslea. A fermentation method is described which allows obtaining an increase in the production of lycopene, based on the addition of trisporic acids. The process is performed starting from wild strains or mutants of them, in single or mixed culture. The addition of trisporic acids can take place at the start of or during fermentation and their origin can be a partially purified preparation or a non-purified source of them.

Description

Lycopene production method.

Field of the Invention

The present invention relates to a new process for the production of carotenoids, especially lycopene, from submerged cultures of mucoral Blakeslea fungi. A fermentation method is described by which it is possible to obtain an increase in lycopene production, based on the addition of extracts enriched in trisporic acids. The process is carried out from wild lines, or mutants thereof, in simple or mixed culture. The addition of trisporic acids can be done at the beginning or during fermentation.

State of the art

Carotenoids are abundant compounds in fruits and vegetables, and have been the subject of numerous studies due to its properties as antioxidants and as precursors of vitamin A. Lycopene, in addition to its antioxidant property, prevents cardiovascular diseases and some types of cancer such as prostate cancer and gastrointestinal cancer and acts in the growth control (Stahl W. and Sies, H. 1996. Arch. Biochem. Biophys 336: 1-9; Clinton, SK 1998. Nutr. Rev. 56: 35-51). The carotenoids, due to their coloration yellow to red, they are also used as a supplement and dye food of margarine, butter, salad oils, soups and sauces (Ninet L. and Renaut J. 1979. In: Peppler HJ., Perlman D. (eds). Microbial Technology, 2nd Edn., Vol. 1 Academic Press, NY, pp. 529-544).

Lycopene is a carotenoid that is produced as intermediary in the biosynthetic route of β-carotene (GB 1034944 and GB 1114043) and xanthophylls.

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one

Lycopene preparations are obtained from tomato (PCT Patents WO 97/48287, EP 608027), or by fermentation of mucoral fungi such as Phycomyces , Blakeslea and Choanephora (Patents GB 1008469, US 3097146, US 3369974, JP 73016189, JP 73016190 and RU 2102416).

GB 1008469 and US 3097146 disclose a method of fermentation of the Blakeslea trispora fungus consisting of a culture medium whose pH is maintained above 7.0 and up to 9.5. After seven days of incubation, 9.97 mg of lycopene is obtained per 100 ml of medium.

Japanese patents JP 73016189 and JP 73016190 describe a method of fermentation with mucoral fungi such as Phycomyces , Blakeslea and Choanephora , in which tertiary amines are added to prevent the formation of β-carotene and favor the accumulation of lycopene. As an example of tertiary amines, the following are mentioned: hordenine, 2-dimethylamino-ethanol; 3-dimethylamino-1-propanol; 1-dimethylamino-2-propanol; 2-diethylamino-ethanol; 3-diethylamino-1-propanol; 1-diethylamino-2-propanol; triethylamine; 2-diisopropylamino-ethanol; 3-diisopropylamino-1-propanol; 1-diisopropylamino-2-propanol; 2-di-n-propylamino-ethanol; 3-di-n-propylamino-1-propanol; 1-di-n-propylamino-2-propanol; 2-dimethylaminoethylbenzene; 2-diethylaminoethylbenzene; p- (2-diethylaminoethyl) phenol. RU 2102416 describes the addition of aminomethylpyridines and tobacco residues to induce the accumulation of lycopene. In addition to the substances described in said patents, the use of other heterocyclic nitrogenous bases capable of blocking the synthesis of carotenoids at the lycopene level, such as nicotine (Japanese patent JP 09313167), imidazole, pyridine, morpholine, quinoline and some derivatives has been described. substituted (US Patent 3369974; Ninet L., Renaut J. 1979. In: Peppler HJ, Perlman D (eds.). Microbial Technology, 2nd Ed., Vol. 1 Academic Press, NY, pp. 529-544).

B. trispora mutants that accumulate lycopene without the need to add tertiary amines have also been described (Mehta B. and Cerdá-Olmedo E. 1995. Appl. Microbiol. Biotechnol. 42: 836-838).

In any of the methods of production of β-carotene with the fungus B. trispora mixed cultures of the fungus are used, as a result of which yields of β-carotene are obtained far superior to those found in the (+) and (-) separately (Ciegler, A. 1965. Advances in Applied Microbiology 7: 1-34; Plempel, M. 1965. Plant 65: 225-231; Sutter, RP. and Rafelson, ME. 1968. J. Bacteriology 95 : 426-432). The increase in β-carotene production in mixed cultures is correlated with the production of a family of acid compounds called β factor or trisporic acids. (Caglioti L., Cainelli G., Camerino B., Mondelli R., Prieto A., Quilico A., Salvatori T, Selva A. 1966. Tetrahedron Supplement 7: 175-187). It has been proposed that trisporic acids are sex hormones in mucoral fungi such as B. trispora , Phycomyces blakesleanus and Mucor mucedo , since in addition to stimulating the production of β-carotene, they stimulate the formation of zygophores (van den Ende, H. 1968 J. Bacteriology 96: 1298-1303; Austin DJ., Bu'Lock JD., Gooday GW 1969. Nature 223: 1178-1179; Reschke, T. 1969. Tetrahedron Letters 29: 3435-3439; van den Ende H. , Wiechmann AHCA., Reyngoud DJ., Hendricks T. 1970. J. Bacteriology 101: 423-428).

Chemically, trisporic acids are oxidized and unsaturated derivatives of 1,1,3-trimethyl-2- (3-methyloctyl) cyclohexane. Said compounds can be classified as processed sesquiterpenes or degradation products of diterpenes or carotenoids. Trisporic acids have been characterized as a mixture of three substances: trisphoric acid A, B and C, also called factor? 1,? 2 and? 3 (because they stimulate the synthesis of? beta-carotene). The most abundant is trisphoric acid C, which is present in 80% in mixed cultures of B. trispora (Caglioti, L., Cainelli, G., Camerino, B., Mondelli, R., Prieto, A., Quilico, A., Salvatori, T, Selva, A. 1966. Tetrahedron Supplement 7: 175-187).

2

Trisporic acids are synthesized from β-carotene produced by both the strain (+) as per the lineage (-). The β-carotene is Retinal metabolized via 4-hydrotrisporol. This compound is metabolized by lineage (+) in acid 4-dihydrotrisphoric and in its methyl ester, and the line (-) converts 4-hydrotrisporol into trisporol These two intermediaries are converted to acid trisphoric only after spreading in the middle until the lineage of opposite sex (Compiled by Gooday GW. and Carlile MJ. 1997. Mycologist 11: 26-30). That is, the lineage (-) produces the intermediates that are converted into trisphoric acid by the line (+) and vice versa, according to the previous scheme.

Trisporic acids obtained from mixed cultures of B. trispora can be used to increase the production of β-carotene in cultures with independent strains. For example, the addition of 21.8 units of factor b increases by 422% the yield of β-carotene from the lineage (-) and by 71% the yield of the lineage (+). However, the stimulation of carotenogenesis per unit of β factor in lineage cultures (-) was only 20-30% of the carotenogenesis produced in mixed cultures and with similar amounts of β factor. (Sutter RP. And Rafelson ME. 1968. J. Bacteriology 95: 426-432).

Purification of trisporic acids can be performed by various methods, such as those described by Sutter, R. 1970. Science 168: 1590-1592 or Sutter RP., Capage DA., Harrison TL., Keen WA. 1973. J. Bacteriology 114: 1074-1082.

It is not known that an effect has been described positive increase in lycopene yield by means of Addition of trisporic acids. Lycopene production is based on suppress lycopene cyclase activity that converts lycopene into β-carotene, which results in low levels of β-carotene in the fermentation. Trisporic acids, which are sex hormones that induce carotenogenesis, are synthesized from β-carotene. It is therefore the absence of precursor (β-carotene) which inhibits the formation of these hormones and the one that allows to increase the lycopene production through the use of trisporic acids, since in mixed cultures that produce β-carotene (as in the case of mutant SB34 (-) with ASA2 (+) of examples 5 and 6), the addition of trisporic acids has no effect.

Detailed description of the invention

The object of the present invention aims to increase the production of carotenoids, especially lycopene, in a fermentation process with mucoral fungi ( Blakeslea, Choanephora or Phycomyces ), more specifically in B. trispora . The invention consists in culturing the B. trispora fungus in a fermentation medium and adding partially purified (or PTA) or non-purified (PTA) or trisporic acids (NPTA) from any other source, such as a medium for β-carotene production.

The effect of trisporic acids on Lycopene fermentation is based on the fact that such hormones are synthesized from β-carotene, a compound that cannot be formed in Lycopene fermentation. Under these conditions the fungus lacks precursor for the synthesis of these hormones and carotenogenesis It is not induced to its maximum degree.

The organisms used for fermentation can be isolated strains or a mixture of the (+) and (-) strains of mucoral fungi, more specifically B. trispora , or alternatively mutants of B. trispora capable of accumulating lycopene. Various mixtures of (+) and (-) line of the fungus can be used in the fermentation process of this invention. In the case of using strains of B. trispora that are producers of β-carotene, compounds are added that are capable of blocking carotenogenesis at the lycopene level (for example tertiary amines and others mentioned above). In the case of using mutants blocked in the biosynthesis of carotenoids from lycopene, the addition of tertiary amines and related compounds will not be necessary. In either of the two cases cited, trisporic acids are added partially purified (PTA) or unpurified (NPTA) from some source thereof, such as from culture medium from a fermentation of β- carotene, and in both cases at an approximate concentration of 0.35 mg / ml. The term NPTA corresponds to a β-carotene medium. In Fig. 1, the concentration in said media of trisporic acids can be calculated according to the incubation day (on the 4th day it would be between 350-400 µg / ml).

The process for the production of carotenoids from mucoral fungi, and more specifically lycopene with selected strains or mutants of B. trispora , can be carried out in any culture medium that contains one or more carbon sources, one or more nitrogen sources. , mineral salts and thiamine. Carbon sources can be added as simple or complex nutrients and include carbohydrates or fats, such as dextrins, starches, glucose, sucrose, fructose, animal or vegetable oils. The culture medium must also carry an assimilable source of nitrogen, organic or inorganic, such as soybean meal, cornmeal, soluble distillates, yeast extract, cottonseed meal, peptone, casein, ammonium sulfate, etc. Among the mineral salts that can be added to the culture medium are phosphates, sulfates, chlorides, sodium, potassium, ammonium, calcium and magnesium. Nutrient ratios are determined based on the growth needs of the microorganism and production levels. Fermentation is preferably carried out under aerobic conditions and in submerged culture. The fermentation temperature may range between 20 and 32 ° C, although between 25 and 28 ° C is preferred.

The addition of trisporic acids in a concentration ranging from 0.001-1000 mg / ml it can be made from a partially purified preparation or from any source thereof, such as from culture medium from a fermentation of β-carotene of any mucoral fungus. The Addition is preferably done between 72-144 h of the beginning of fermentation.

Lycopene is extracted by any described method of cell rupture that allows to release the intracellular content, which dissolves in any solvent in whichever is soluble. Its concentration can be determined by spectrophotometric measurement, but the use of chromatography is preferred liquid (HPLC), by any of the methods described in the bibliography.

The lines used are the following: β-carotene producing mutants: ASA2 (+) and ASA25 (-). Lycopene mutant SB34 (-). The latter mutant also produces 25% of β-carotene (see Table 1). The selection of mutants is irrelevant from the point of view of the effect of increased carotene production due to trisporic acids per se . That is, the effect due to said compounds would be reproducible in any other analogous mutant strain, even if it had a lower basal lycopene production, as is the case with parental strains, for example. The parental strains are deposited in a collection and are viable and accessible to the public.

Example 1 Determination of the concentration of trisporic acids at throughout a fermentation of β-carotene and of lycopene

An inoculum medium is prepared containing per liter: soy flour, 23 g; cornmeal, 47 g; 0.5 g monopotassium phosphate; thiamine hydrochloride, 0.002 g. Its initial pH is 6.3. The medium is distributed in 500 ml Erlenmeyer flasks at a rate of 67 or 100 ml. After sterilization, strains B. trispora ASA2 (+) and B. trispora ASA25 (-) are seeded from spore suspensions in separate flasks and incubated at 25 ° C for 48 hours.

The mutant strains B. trispora ASA2 (+) and B. trispora ASA25 (-) are obtained by mutation with NTG from the wild strains of B. trispora F208 (-) and F117 (+) deposited in the Collection of Industrial Microorganisms (VKM), located in Moscow, Russia, both strains being accessible to the public.

A base fermentation medium is prepared that Contains per liter: soy flour, 44 g; cornmeal, 19 g; dibasic phosphate 0.55 g; thiamine hydrochloride, 0.002 g; oil 10% vegetable. Its initial pH is adjusted to 7.5 with potassium hydroxide. The medium is distributed in 250 ml Erlenmeyer flasks at a rate of 20 ml.

Flasks containing 10% of a mixed culture of the B. trispora ASA2 (+) and B. trispora ASA25 (-) strains are inoculated for the fermentation of β-carotene. For the fermentation of lycopene a chemical agent is added to the base medium in order to block the biosynthetic pathway at the lycopene level, such as 2 ml / l of pyridine. This medium is inoculated with 10% of a mixed culture of the B. trispora ASA2 (+) and B. trispora ASA25 (-) lines. All flasks are incubated at 25 ° C on an orbital shaker at 250 rpm. From the second day of fermentation a sample is taken every 24 hours and the concentration of trisporic acids in β-carotene producing flasks and in lycopene producing flasks is determined.

The determination of acid concentration Trisporic is carried out by extraction with a volume of chloroform after acidifying the sample at pH 2. The fraction Organic is extracted with a volume of a bicarbonate solution 4% sodium, the aqueous phase is collected, which is acidified to extract it again with chloroform. Subsequently the chloroform is evaporated to dryness and the residue consisting of acids Trisporic is dissolved in ethanol or buffer Tris sulfate. Trisporic acids are quantified by absorption spectrometry at 325 nm, assuming a absorptivity of 70 ml x mg -1 - cm -1 - (Sutter RP., Capage DA., Harrison TL., Keen WA. 1973. J. Bacteriology 114: 1074-1082).

The concentration of trisporic acids was be different throughout the fermentation of β-carotene and lycopene, being significantly lower in the latter. The maximum difference is found on the 4th day, with a concentration of 378 µg / ml in the fermentation of β-carotene and 46 µg / ml in lycopene fermentation (an 8-fold difference approximately) (Fig. 1).

Example 2 Effect of the addition of trisporic acids on fermentation of a mutant partially blocked in the biosynthesis of β-carotene in a way that accumulates lycopene

The inoculum medium is prepared in the manner described in Example 1. After sterilization, strain B. trispora SB34 (-) is seeded from a spore suspension and incubated at 25 ° C for 48 hours.

The mutant strain B. trispora SB34 (-) was obtained by mutation with NTG from the wild strain B. trispora F921 (-) deposited in the VKM crop collection, Moscow, Russia and accessible to the public.

A fermentation medium of the same type as described in Example 1 is prepared. After sterilization, a series of flasks are inoculated with 10% of the B. trispora SB34 (-) line, characterized by being a partially blocked mutant in the biosynthesis of β-carotene so that it accumulates lycopene. All flasks are incubated at 25 ° C on an orbital shaker at 250 rpm.

Control flasks are those that do not receive Addition of trisporic acids. The flask group that receives Trisporic acid addition remains in the same conditions that the control until the 4th day, when a preparation is added enriched in trisporic acids, purified as described in the Example 1, at a final concentration of 350 µg / ml.

With the addition of partially trisphoric acids purified (PTA) an increase of 0.11 to 0.24 mg of lycopene per gram of dry weight (an increase of 2.18 times with regarding the control on the 6th day of fermentation, Fig. 2).

Example 3 Effect of the addition of trisporic acids on fermentation of lycopene with a mutant producing β-carotene whose biosynthesis is blocked at the level of lycopene by a chemical agent

The inoculum medium is prepared in the manner described in Example 1. After sterilization, strain B. trispora ASA25 (-) is seeded from a spore suspension and incubated at 25 ° C for 48 hours.

A fermentation medium of the same type as described in Example 1 is prepared. After sterilization, a chemical agent that blocks the biosynthesis of carotenoids at the level of lycopene, such as imidazole at a final concentration of 0.75 mg, is added / ml This medium is also supplemented with pH 7.5 phosphate buffer at a final concentration of 25 mM and inoculated with 10% of the B. trispora ASA25 (-) line. The flasks are incubated at 25 ° C on an orbital shaker at 250 rpm.

Control flasks are those that do not receive Addition of trisporic acids. The flask group that receives Trisporic acid addition remains in the same conditions that the control until the 4th day, when a preparation is added enriched in trisporic acids at a final concentration of 350 \ mug / ml. The purification of trisporic acids is carried out by same way described in Example 1.

With the addition of partially trisphoric acids purified (PTA) an increase of 0.16 to 1.09 mg of lycopene per gram of dry weight (an increase of 6.8 times with regarding the control on the 6th day of fermentation, Fig. 3).

Example 4 Effect of the addition of trisporic acids, partially purified (PTA) or unpurified (NPTA), on the fermentation of the B. trispora ASA25 (-) mutant in mixed culture with B. trispora ASA2 (+)

The inoculum medium is performed in the manner described in Example 1. After sterilization, the B. trispora ASA25 (-) and B. trispora ASA2 (+) strains are seeded from spore suspensions in separate flasks and incubated at 25 ° C for 48 hours.

A fermentation medium of the same type as described in Example 1 is prepared. After sterilization, imidazole is added at a final concentration of 0.75 mg / ml and phosphate buffer pH 7.5 at a final concentration of 25 mM . This medium is inoculated with 10% of a mixture of the B. trispora ASA25 (-) and B. trispora ASA2 (+) lines. The flasks are incubated at 25 ° C on an orbital shaker at 250 rpm.

To obtain a source of unpurified trisporic acids (NPTA), a fermentation medium of the same type as described in Example 1 is prepared. After sterilization, a mixed culture is planted with 10% of the B. trispora ASA2 strains . (+) and B. trispora ASA25 (-), which are producers of β-carotene. The flasks are incubated at 25 ° C on an orbital shaker at 250 rpm.

Control flasks are those that receive no addition of trisporic acids. The group of flasks that receive the addition of partially purified trisporic acids (PTA), is maintained in the same conditions as the control until day 4, in which trisporic acids are added at a final concentration of 350 µg / ml. The purification of trisporic acids is carried out in the same manner described in Example 1. The flask group that receives the addition of unpurified trisporic acids (NPTA) is maintained in the same conditions as the control until the 4th day, to be then subjected to a filtration process through a 20 mm nylon filter that allows to separate the mycelium from the B. trispora ASA25 (-) and B. trispora ASA2 (+) strains from the culture medium where they have grown in the presence of imidazole. In parallel, the separation of mycelium and culture medium is carried out through a 20 µm nylon filter, but in this case of flasks containing the two strains B. trispora ASA2 (+) and B. trispora ASA25 (-), under conditions of β-carotene production, whose culture medium is considered an unpurified source of trisphoric acids (NPTA). After the separation of mycelium and culture medium, the mycelium of the B. trispora ASA25 (-) and ASA2 (+) strains grown with imidazole is mixed with the culture medium of the same strains grown in the absence of imidazole. Imidazole is added to the culture medium considered as an unpurified source of trisphoric acids (NPTA) to obtain a final concentration of 0.75 mg / ml. These flasks are incubated at 25 ° C and 250 rpm.

With the addition of partially trisphoric acids purified (PTA) an increase of 29.14 to 43.58 mg of lycopene per gram of dry weight (an increase of 50% over at the 6th day fermentation control, Fig. 3) and by adding of β-carotene culture medium is achieved an increase of 29.14 to 41.07 mg of lycopene per gram of dry weight (an increase of 41% compared to the control on the 6th day of fermentation, Fig. 4). This example demonstrates that the effect of Trisporic acids is independent of their degree of purification.

Example 5 Effect of the addition of trisporic acids on the fermentation of the B. trispora SB34 (-) mutant in mixed culture with B. trispora ASA2 (+)

The inoculum medium is prepared in the manner described in Example 1. After sterilization, strains B. trispora SB34 (-) and B. trispora ASA2 (+) are seeded from spore suspensions in separate flasks and incubated at 25 ° C for 48 hours.

A fermentation medium of the same type as described in Example 1 is prepared. After sterilization it is inoculated with 10% of a mixture of the B. trispora SB34 (-) and B. trispora ASA2 (+) strains. The flasks are incubated at 25 ° C on an orbital shaker at 250 rpm.

Control flasks are those that do not receive Addition of trisporic acids. The flask group that receives Trisporic acid addition remains in the same conditions that the control until the 4th day, when a preparation is added enriched in trisporic acids at a final concentration of 350 \ mug / ml. The purification of trisporic acids is carried out by same way described in Example 1.

The addition of a preparation enriched in Trisporic acids has no significant effect on production lycopene when the fermentation of the SB34 (-) mutant is carried out in mixed culture (Fig. 5).

Example 6 Analysis of the proportion of carotenoids in mixed fermentations of strains B. trispora SB34 (-) or B. trispora ASA 25 (-) with B. trispora ASA2 (+)

The inoculum medium is prepared in the manner described in Example 1. After sterilization, strains B. trispora SB34 (-), B. trispora ASA25 (-) and B. trispora ASA2 (+) are seeded from spore suspensions. ) in separate flasks and incubate at 25 ° C for 48 hours.

A fermentation medium of the same type as described in Example 1 is prepared. After sterilization, this medium is divided into two groups. A group of flasks is inoculated with 10% of a mixture of the B. trispora SB34 (-) and B. trispora ASA2 (+) strains. To the other group of flasks, after sterilization, imidazole is added at a final concentration of 0.75 mg / ml and phosphate buffer pH 7.5 at a final concentration of 25 mM. This medium is inoculated with 10% of a mixture of the B. trispora ASA25 (-) and B. trispora ASA2 (+) lines. All flasks are incubated at 25 ° C on an orbital shaker at 250 rpm.

Control flasks are those that do not carry Addition of trisporic acids. The flask group that receives Trisporic acid addition remains in the same conditions that the control until the 4th day, when a preparation is added enriched in trisporic acids at a final concentration of 350 \ mug / ml. The purification of trisporic acids is carried out by same way described in Example 1.

Sample of the cultures is collected on the 5th and 6th day of fermentation in order to analyze the proportion of carotenoids by liquid chromatography (HPLC). The results obtained are shown in Table 1.

TABLE 1 Percentage of carotenoids on the 5th and 6th day of fermentation in mixed culture of the B. trispora SB34 (-) or ASA25 (-) strains with B. trispora ASA2 (+)

Strains Acids trisporic Day Neurosporene Lycopene γ-carotene β-carotene (%) (%) (%) (%) SB34 (-) xASA2 (+) Do not 5th 3 57 16 25 SB34 (-) xASA2 (+) Do not 6th 3 64 12 twenty-one SB34 (-) xASA2 (+) Yes 5th 3 55 fifteen 28 SB34 (-) xASA2 (+) Yes 6th 2 62 12 2. 3 ASA25 (-) xASA2 (+) Do not 5th one 88 9 2 ASA25 (-) xASA2 (+) Do not 6th one 89 8 2 ASA25 (-) xASA2 (+) Yes 5th one 85 10 3 ASA25 (-) xASA2 (+) Yes 6th one 88 9 2

The analysis of the percentage of carotenoids obtained in the fermentation of lycopene using a mixed culture of the B. trispora SB34 (-) or B. trispora ASA25 (-) with B. trispora ASA2 (+) lines shows a significant difference in the percentage of lycopene and β-carotene (Table 1). These differences are independent of the addition of trisporic acids. The mixture of carotenoids obtained on the 6th day by fermentation with B. trispora SB34 (-) has 63% lycopene, while that obtained with B. trispora ASA25 (-) has 88% (yielding a product that is 25% more pure). In the same mixture, the proportion of β-carotene is 10 times higher in fermentation with B. trispora SB34 (-) than with B. trispora ASA25 (-).

Since β-carotene is the precursor of trisporic acids, these data could explain:

one.

The difference in concentration of trisporic acids between a β-carotene fermentation and fermentation of lycopene (Example 1).

2.

The fact that the addition of trisporic acids (purified or unpurified) increases lycopene yield by 40 to 50% on the 6th day of fermentation in mixed culture when used as a negative strain B. trispora ASA25 (-), whose mixture of carotenoids contains only 2% of β-carotene (Examples 4 and 6).

3.

The fact that the addition of trisporic acids has no effect on the mixed culture when used as a negative strain B. trispora SB34 (-), whose carotenoid mixture contains more than 20% of β-carotene (Examples 5 and 6).

These results demonstrate that the effect of trisporic acids on lycopene fermentation in crops mixed is due to the absence of precursor for its biosynthesis. From there that its use in the industrial production of lycopene from High purity is highly advantageous.

Detailed description of the figures

Fig. 1. Concentration of trisporic acids along a β-carotene fermentation and a lycopene fermentation. In both fermentations a mixed culture of the B. trispora ASA25 (-) and B. trispora ASA2 (+) lines is used. In the fermentation of lycopene (squares) a chemical agent is added to the culture medium that favors the accumulation of lycopene, in this case pyridine. The fermentation of β-carotene (rhombuses) is carried out under the same conditions but without pyridine. Abscissa: time in days. Sorted: concentration of trisporic acids (mug / ml).

Fig. 2. Production of lycopene by mutant strain B. trispora SB34 (-), blocked in the synthesis of β-carotene at the lycopene level, with and without the addition of trisporic acids. The lycopene production values obtained in a control fermentation without the addition of trisporic acids are represented by rhombuses. The effect of the addition of partially purified trisporic acids (PTA) on the 4th day of fermentation on lycopene production is indicated by squares. The values represent the average of two independent samples. Error bars represent the standard deviation of the average. Abscissa: time in days. Sorted: mg lycopene / g dry weight.

Fig. 3. Production of lycopene by mutant strain B. trispora ASA25 (-), superproducer of β-carotene, with and without the addition of trisporic acids. The fermentation medium is supplemented with imidazole to favor the accumulation of lycopene. The lycopene production values obtained in a control fermentation without the addition of trisporic acids are represented by rhombuses. The effect of the addition of partially purified trisporic acids (PTA) on the 4th day of fermentation on lycopene production is indicated by squares. The values represent the average of two independent samples. Error bars represent the standard deviation of the average. Abscissa: time in days. Sorted: mg lycopene / g dry weight.

Fig. 4. Production of lycopene in mixed culture of the superproductive mutant strains of β-carotene B. trispora ASA25 (-) and B. trispora ASA2 (+), with and without the addition of trisporic acids. The fermentation medium is supplemented with imidazole to favor the accumulation of lycopene. The lycopene production values obtained in a control fermentation without the addition of trisporic acids are represented by rhombuses. The effect of the addition of partially purified trisporic acids (PTA) on the 4th day of fermentation on lycopene production is indicated by squares. The lycopene production obtained by adding unpurified trisporic acids (NPTA) on the 4th day of fermentation is represented by circles. The values represent the average of two independent samples. Error bars represent the standard deviation of the average. Abscissa: time in days. Sorted: mg lycopene / g dry weight.

Fig. 5. Production of lycopene in mixed culture of the B. trispora SB34 (-) and B. trispora ASA2 (+) strains, with and without the addition of trisporic acids. The lycopene production values obtained in a control fermentation without the addition of trisporic acids are represented by rhombuses. The effect of the addition of partially purified trisporic acids (PTA) on the 4th day of fermentation on lycopene production is indicated by squares. The values represent the average of two independent samples. Error bars represent the standard deviation of the average. Abscissa: time in days. Sorted: mg lycopene / g dry weight.

Claims (8)

1. A process for the production of lycopene by fermentation of (+) and (-) mucoral fungi under inhibitory conditions to produce β-carotene, characterized in that trisporic acids are added at any time during fermentation and the lycopene produced is recovered . 2. A method according to claim 1, characterized in that the mucoral fungus used in fermentation is Blakeslea trispora . 3. A method according to any one of claims 1 and 2, characterized in that a culture containing a mutant strain of B. trispora is used whose biosynthesis capacity of β-carotene is totally or partially blocked, due to the mutation, and which accumulates lycopene 4. A method according to any one of claims 1 and 2, characterized in that an inhibitor of β-carotene biosynthesis is added to the culture, at any time during fermentation, to induce the accumulation of lycopene. 5. A process according to any of the preceding claims, characterized in that the addition of trisporic acids is carried out in such a way that a concentration thereof ranging between 0.001 and 1000 mg / ml is achieved. 6. A process according to any of the preceding claims, characterized in that the addition of trisporic acids to the culture occurs between 72 and 144 h from the beginning of the fermentation. 7. A process according to any one of the preceding claims, characterized in that the trisporic acids are added partially purified. A process according to any one of claims 1 to 6, characterized in that the trisporic acids are added without purification, preferably from a culture medium recovered from a β-carotene fermentation.